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- EMDB-2965: Cryo-EM structure of Ross River virus -

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Basic information

Entry
Database: EMDB / ID: EMD-2965
TitleCryo-EM structure of Ross River virus
Map dataCryo-EM 3D reconstruction of Ross River virus
Sample
  • Sample: Cryo-EM structure of Ross River virus
  • Virus: Ross River virus
KeywordsRoss River virus
Biological speciesRoss River virus
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 15.2 Å
AuthorsChen C / Wang J C-Y / Rayaprolu V / Mukhopadhyay S / Zlotnick A
CitationJournal: ACS Nano / Year: 2015
Title: Self-Assembly of an Alphavirus Core-like Particle Is Distinguished by Strong Intersubunit Association Energy and Structural Defects.
Authors: Joseph Che-Yen Wang / Chao Chen / Vamseedhar Rayaprolu / Suchetana Mukhopadhyay / Adam Zlotnick /
Abstract: Weak association energy can lead to uniform nanostructures: defects can anneal due to subunit lability. What happens when strong association energy leads to particles where defects are trapped? ...Weak association energy can lead to uniform nanostructures: defects can anneal due to subunit lability. What happens when strong association energy leads to particles where defects are trapped? Alphaviruses are enveloped viruses whose icosahedral nucleocapsid core can assemble independently. We used a simplest case system to study Ross River virus (RRV) core-like particle (CLP) self-assembly using purified capsid protein and a short DNA oligomer. We find that capsid protein binds the oligomer with high affinity to form an assembly competent unit (U). Subsequently, U assembles with concentration dependence into CLPs. We determined that U-U pairwise interactions are very strong (ca. -6 kcal/mol) compared to other virus assembly systems. Assembled RRV CLPs appeared morphologically uniform and cryo-EM image reconstruction with imposed icosahedral symmetry yielded a T = 4 structure. However, 2D class averages of the CLPs show that virtually every class had disordered regions. These results suggested that irregular cores may be present in RRV virions. To test this hypothesis, we determined 2D class averages of RRV virions using authentic virions or only the core from intact virions isolated by computational masking. Virion-based class averages were symmetrical, geometric, and corresponded well to projections of image reconstructions. In core-based class averages, cores and envelope proteins in many classes were disordered. These results suggest that partly disordered components are common even in ostensibly well-ordered viruses, a biological realization of a patchy particle. Biological advantages of partly disordered complexes may arise from their ease of dissociation and asymmetry.
History
DepositionMar 26, 2015-
Header (metadata) releaseMay 6, 2015-
Map releaseAug 26, 2015-
UpdateSep 30, 2015-
Current statusSep 30, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.039
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.039
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2965.png

Downloads & links

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Map

FileDownload / File: emd_2965.map.gz / Format: CCP4 / Size: 319.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM 3D reconstruction of Ross River virus
Voxel sizeX=Y=Z: 1.84 Å
Density
Contour LevelBy AUTHOR: 0.039 / Movie #1: 0.039
Minimum - Maximum-0.19181091 - 0.18434374
Average (Standard dev.)0.00858032 (±0.03060816)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-220-220-220
Dimensions441441441
Spacing441441441
CellA=B=C: 811.44 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.841.841.84
M x/y/z441441441
origin x/y/z0.0000.0000.000
length x/y/z811.440811.440811.440
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-220-220-220
NC/NR/NS441441441
D min/max/mean-0.1920.1840.009

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Supplemental data

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Sample components

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Entire : Cryo-EM structure of Ross River virus

EntireName: Cryo-EM structure of Ross River virus
Components
  • Sample: Cryo-EM structure of Ross River virus
  • Virus: Ross River virus

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Supramolecule #1000: Cryo-EM structure of Ross River virus

SupramoleculeName: Cryo-EM structure of Ross River virus / type: sample / ID: 1000
Details: The virus was purified from Baby Hamster Kidney cells
Number unique components: 1

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Supramolecule #1: Ross River virus

SupramoleculeName: Ross River virus / type: virus / ID: 1 / NCBI-ID: 11029 / Sci species name: Ross River virus / Virus type: VIRION / Virus isolate: OTHER / Virus enveloped: Yes / Virus empty: No
Host (natural)Organism: Aedes (mosquito) / synonym: INVERTEBRATES
Host systemOrganism: Mesocricetus auratus (golden hamster) / Recombinant cell: BHK-21 / Recombinant plasmid: cDNA clone of Ross River T48 virus
Virus shellShell ID: 1 / T number (triangulation number): 4

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4 / Details: 20 mM Hepes, 150 mM NaCl and 0.1 mM EDTA
StainingType: NEGATIVE / Details: cryo-EM
GridDetails: 300 mesh copper grid with thin carbon support
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III / Method: wait 25 sec and blot for 4 sec before plunging

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Electron microscopy

MicroscopeJEOL 3200FS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.1 mm / Nominal defocus max: 4.01 µm / Nominal defocus min: 1.31 µm / Nominal magnification: 60000
Specialist opticsEnergy filter - Name: omega filter / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder: 626 / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 100 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 60,000 times magnification
DetailsMDS
DateMar 15, 2015
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 127 / Average electron dose: 25 e/Å2

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Image processing

CTF correctionDetails: per micrograph
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.2 Å / Resolution method: OTHER / Software - Name: auto3dem / Number images used: 6146
DetailsThe particles were selected using e2boxer.py and the images were processed using auto3dem. The initial model was built de novo.

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